NEW YORK – A new method can capture mRNA that is being translated by ribosomes throughout tissues, with potential to help elucidate much of RNA biology from transcription to degradation.
Last week, researchers from MIT and the Broad Institute led by Xiao Wang published a study in Science describing ribosome-bound mRNA mapping (Ribomap), which provides information on translation in situ, in 3D, and with single-cell resolution. Their targeted sequencing assay detects ribosome-bound mRNAs using a set of three probes to produce barcoded DNA amplicons in tissues. The barcodes are then read out through an in situ sequencing method.
Ribomap can profile hundreds to thousands of transcripts as they're being translated into proteins. In a proof-of-concept experiment, the team used it to monitor the translation of 981 genes in single HeLa cells and identified mRNAs that are translated at different stages of the cell cycle. Further, in an experiment using mouse brain tissue, they were able to measure and map the translation of 5,413 genes across more than 119,000 cells.
"There have been a lot of fluorescence in situ hybridization (FISH)- or sequencing-based spatial transcriptomics methods, but most of them are just looking at gene expression," said Rong Fan, a Yale University researcher who has developed spatial biology technologies and who wrote an accompanying commentary for Science. "Every single RNA molecule has an interesting life cycle. There has not really been any technology that allows you to look at that, at this scale."
While the new paper focuses on mRNA translation, the tri-probe approach is "pioneering" and "very versatile," Fan said, and could be used to study other stages of the RNA life cycle. "For example, mRNA degradation rate might be studied by detecting endonuclease-bound mRNA molecules," he said.
The method offers improved scale and resolution at a spot with a critical coverage gap in molecular biology. "Everything [after] DNA expression was basically ignored by previous research," said Jingyi "Rena" Ren, a graduate student in Wang's lab and a lead author on the paper.
Moreover, messenger RNA and protein levels are poorly correlated, so spatial profiling of mRNAs in the process of translation might provide a closer measure of protein expression. "Indeed, we showed examples in mouse brain data that Ribomap has better correlation with protein than the transcriptome data," said Hu Zeng, a postdoc in Wang's lab and another lead author.
Ribomap builds on recent success in spatial transcriptomics methods, some of which also offer information on RNA dynamics. It uses spatially-resolved transcript amplicon readout mapping (STARMap), an in situ RNA sequencing method developed by Wang while she was a postdoc in Karl Deisseroth's lab at Stanford University.
Ribomap is not the first tool developed for mRNA translation analysis, but it offers two distinct advantages over an earlier method that fixed and precipitated ribosome-bound mRNAs for analysis with next-generation sequencing: higher throughput and the addition of spatial information.
To prepare samples for reading with STARMap, the method uses three probes. A padlock probe targets mRNA molecules of interest and adds a gene-specific barcode. Another probe binds to the padlock probe and serves as a primer for rolling circle amplification. This amplification step relies on the presence of a third probe, which is hybridized to 18S rRNA inside ribosomes and binds to the padlock probe, acting as a splint to help circularize it. Thus, only actively translating mRNAs are analyzed.
Fan stressed that STARMap is used only to read out the barcodes introduced by the padlock probes and not the sequence of the translating RNAs. But a true sequencing of these RNAs could be possible. "I would love to see one day people adopt this tri-probe approach to do truly base-by-base sequencing of translating RNA or nascent RNA," he said.
That means that despite its impressive multiplexing ability, Ribomap is limited to detecting a finite set of known mRNA molecules, Fan wrote.
While the method has not yet been adapted to work in human tissues, that's a focus of the lab, Ren said. "We'll probably use it to look at other kinds of tissues and organs and do some spatial atlas-type of work," she said.
Fan said Ribomap's tri-probe design could be adapted to work in many other areas of RNA research. The authors disclosed that they have applied for patents related to the technology and Wang said Stellaromics, a firm she cofounded, is in the process of licensing Ribomap from the Broad. "We are also spinning off STARmap/Ribomap internally at Broad to enable more collaborations and provide services," she said in an email. Outside of the Wang and Deisseroth labs, at least three other US labs run STARMap, she said.
"I think this approach can be applied to even existing FISH-based technologies," Fan said, including 10x Genomics' Xenium in situ analyzer and Resolve Bioscience's probe-based spatial transcriptomics method.
"In the future, the latest breakthroughs in spatial epigenomics and multiomics could be integrated with Ribomap to link RNA translation to epigenetic control, protein expression, and metabolic function," Fan wrote in his article. "Such an approach would span the central dogma of molecular biology, which defines the flow of genetic information from DNA to RNA, to protein and cellular function."